The P2X receptors (P2XRs) are a family of ligand-gated channels activated by extracellular ATP through a sequence of conformational transitions between closed, open, and desensitized states. In this study, we examined the dependence of the activity of P2XRs on ectodomain structure and agonist potency. Experiments were done in human embryonic kidney 293 cells expressing rat P2X(2a)R, P2X(2b)R, and P2X(3)R, and chimeras having the V60-R180 or V60-F301 ectodomain sequences of P2X(3)R instead of the I66-H192 or I66-Y310 sequences of P2X(2a)R and P2X(2b)R. Chimeric P2X(2a)/V60-F301X(3)R and P2X(2b)/V60-F301X(3)R inherited the P2X(3)R ligand-selective profile, whereas the potency of agonists for P2X(2a)/V60-R180X(3)R was in between those observed at parental receptors. Furthermore, P2X(2a)/V60F301X(3)R and P2X(2a)/V60-R180X(3)R desensitized in a P2X(2a)R-specific manner, and P2X(2b)/V60-F301X(3)R desensitized with rates comparable with those of P2X(2b)R. In striking contrast to parental receptors, the rates of decay in P2X(2a)/V60-F301X(3)R and P2X(2b)/V60-F301X(3)R currents after agonist withdrawal were 15- to 200-fold slower. For these chimeras, the decays in currents were not dependent on duration of stimuli and reflected both continuous desensitization and deactivation of receptors. Also, participation of deactivation in closure of channels inversely correlated with potency of agonists to activate receptors. The delay in deactivation was practically abolished in P2X(2a)/V60-R180X(3)R-expressing cells. However, the recovery from desensitization of P2X(2a)/V60-F301X(3)R and P2X(2a)/V60-R180X(3)R was similar and substantially delayed compared with that of parental receptors. These results indicate that both ectodomain halves participate in gating, but that the C and N halves influence the stability of open and desensitized conformation states, respectively, which in turn reflects on rates of receptor deactivation and resensitization.